Rediscovery , systematics and proposed Red List status of Ledebouria junnarensis S . S . Rahangdale and S . R . Rahangdale nom . nov . ( Asparagaceae )-an endemic species from the Western Ghats , Maharashtra , India

A new name in the genus Ledebouria Roth is validated for Scilla viridis Blatter & Hallberg [non Scilla viridis (L.) Salisbury].  It is rediscovered after about 85 years of its first and only report.  It is also redescribed on the basis of morphology, anatomy, cytology and assigned the name Ledebouria junnarensis S.S. Rahangdale & S.R. Rahangdale belonging to the family Asparagaceae, subfamily Hyacinthoideae.  As this is a species endemic to the Western Ghats, Maharashtra, India, it is studied for threat status as per IUCN criteria & guidelines and assigned the status Critically Endangered B1ab(iii)+2ab(iii).  Identification keys for the genera and species of subfamily Hyacinthoideae reported from India are prepared on the basis of reported and observed characters.


INTRODUCTION
The genus Ledebouria Roth of the family Asperagaceae, subfamily Hyacinthoideae is distributed in tropical Africa and Asia including India.The genus was described on the basis of the type species L. hyacinthina Roth from India in 1821 (Venter 2008).The species was later placed in the genus Scilla L. under the section Ledebouria involving many species of genus Scilla by Baker (1870).After the study of South African bulbous Liliaceae collections, Jessop (1970) reinstated the genus Ledebouria, on the basis of bulb with deciduous leaves, erect/flaccid inflorescences, pair of basal ovules in each locule and stipitate ovary.A revision of the genus Ledebouria for South Africa and sub-Saharan collections was done by Venter (2008), comprising more than 60 species.Based on his observations, he stated that, there may be more than one species of this genus in India.Speta (1998a) revised the family Hyacinthaceae and on the basis of morphological, anatomical, developmental, and biochemical characters delimited a total of 65 genera of the family under five subfamilies, viz.: Chlorogaloideae Speta, Oziroeoideae Speta, Urgineoideae Speta, Ornithogloideae Speta and Hyacinthoideae Link.According to her the genus Ledebouria comprising about 50 species and Fessia Speta comprising about 10 species are represented by 1 or 2 species in India, while the genus Scilla L. (sensu stricto) comprising about 30 species is naturally distributed in European, south-west Asian and Mediterranean regions but does not occur in India as well as south of the Sahara.Speta (1998a) also commented that sexual reproduction is predominant; besides this, agamospermy and vegetative reproduction by bulbils may also occur in some taxa.Lenski (1958) reported that, some species of Ledebouria produce bulbils on the leaf tips.The phylogenetic linkages of the taxa in family Hyacinthaceae were studied by Pfosser & Speta (1999) on the basis of plastid DNA sequences.They placed the genus Scilla in Scilla clade, Fessia with two species complexes: (1) the S. hohenackeri Fischer & Meyers group and (2) the S. bisotunensis Speta group in Fessia clade and Ledebouria in Massonieae clade.On the basis of phylogenetic and molecular characters it has been shown that the genus Scilla is not monophyletic and divided into several smaller genera, viz.: Zagrosia Speta, Prospero Salisbury, Othocallis Salisbury, Nectaroscilla Parlatore, Fessia and Scilla (Pfosser & Speta 1999).Further, Stedje (2001) emphasized the use of qualitative morphological characters for generic delimitation of Hyacinthaceae and the molecular or quantitative data as supportive information.Thus, on the basis of the above facts it is clear that the Indian taxa treated under the genus Scilla need critical observations and revision to resolve the taxonomic ambiguity.
Of these, S. viridis Blatt.& Hallb. is endemic to Maharashtra (India) and considered to be 'possibly extinct' (Mishra & Singh 2001).The species was collected for the first time from 'Khandala' [Centre of 'Khandala' Village 18 0 0'N & 73 0 23'E (as per Santapau 1958)] by McCann in vegetative state in September 1918.It was planted in St. Xaviers ' College, Mumbai, where it bloomed in the month of March 1919 and based on this flowering it was described as a new species with the Holotype-14500 (BLAT!) by Blatter & Hallberg (1921).The original protologue of the species comprises only description in the Latin and type specimen, without any figure, geographic coordinates, elevation or ecological notes.Santapau (1958) did not record the species from 'Khandala' in his 'The Flora of Khandala on the Western Ghats of India'.Subsequent attempts by different taxonomists were also unfruitful in collection of the species from Khandala, the type location or elsewhere (Mishra & Singh 2001).Only the Holotype -14500 (BLAT!) is the proof for its occurrence in India (Blatter & Hallberg 1921;Santapau 1958). Therefore, Nayar & Sastry (1987) included it in the Red Data Book under Endangered category and also by Sharma et al. (1996).While doing a reassessment of threatened flowering plants of Maharashtra, Mishra & Singh (2001) were also unable to re-collect the species and it was declared Rediscovery of Ledebouria junnarensis nom.nov.

Rahangdale & Rahangdale
'probably extinct'.The Plant List placed it in the category of 'unresolved taxa names' because of deficiency of information on the taxon and also non-availability of the authentic specimens (The Plant List 2015).
In the year 2005 during the month of June, the present authors collected interesting specimens, which were distinct from commonly occurring Ledebouria revoluta, from Durgawadi-Pimparwadi area of Taluk Junnar, District Pune, Maharashtra State.The vegetative and reproductive characters of the specimens resulted in their identification as S. viridis Blatt.& Hallb.(Rahangdale 2008).Moreover, as there are eco-morphs and cytological variations in L. revoluta, and S. viridis was considered to be possibly extinct, it was decided that, the specimens needed further confirmation on the basis of critical observations, comparison with specimens of other species and detailed study.
The homonymy in the species remained unnoticed for a long time and Karthikeyan et al. (1989), Sharma et al. (1996), and Mishra & Singh (2001) 1 (Ex.1.)and Article 11.4 (For any taxon below the rank of genus, the correct name is the combination of the final epithet of the earliest legitimate name of the taxon in the same rank with the correct name of the genus to which it is assigned).Therefore, a new name is required for the taxon.
As the species has been re-collected after about 85 years since its first and only collection, its IUCN categorization was also needed.Hence, after a critical study during the period 2005 to 2014 with respect to morphology, phenology, anatomy, cytology and reproduction, it was assigned a new name.The IUCN status has also been assigned and the identification keys for the genera and species found in India are prepared and presented in this paper.

Plant Materials
Specimens of the species under study were collected from Durgawadi Pimparwadi area, Taluk Junnar, district Pune, and for comparison the specimens of Ledebouria were also collected from different locations in Maharashtra state comprising the Western Ghats, Konkan, Pune, Marathwada and Vidarbha regions in subsequent years (Table 1).The plant specimens were observed in their natural habitat as well as under cultivation till date for phenology of the specimens, fruit set, and other characters.The herbarium specimens at Blatter Herbarium (BLAT!),St. Xavier's College, Mumbai; Agharkar Herbarium Maharashtra Association for Cultivation of Sciences (AHMA!), Agharkar Research Institute, Pune; and Herbarium of Botanical Survey of India, Western Regional Centre (BSI!), Pune were referred to for comparison and confirmation of identity of the collected specimens.

Methods
The cytological observations were made using root tips treated with an aqueous solution of Colchicine (0.02%) for one hour to arrest the metaphase in dividing root tip cells and in the separation of chromosomes.The root tips were then fixed using Carnoy's Fluid II and then squashed and stained with aceto-carmine.The observations were made under a microscope using 100×10 objective to eyepiece magnification (Dnyansagar 1986).
The observations of anatomical characters were made using Lawrence & Mayo Stereo-zoom microscope for micro-morphological characters and Research microscope attached with micro-photographic set up with a Nikon Coolpix 6000 digital camera.
The identification keys for genera and species recorded from India are prepared on the basis of literature, observed characters of specimens and the online herbarium specimens of the Royal Botanic Gardens Kew, England available on the website www.kew.org/science-conservation/collections/herbarium at apps.kew.org/herbcat/navigator.do.(RBG Kew 2015).
For threat status assessment, the number of individuals was recorded at each visit to the location and the geo-coordinates were obtained using hand-held GPS (Garmin etrex vista HCX).The primary and secondary information required as per IUCN Red List Categories and Criteria version 6.2 were collected, following the guidelines for assigning the criteria (Standards and Petitions Working Group IUCN 2006).

RESULTS
The present study leads to the identification of the specimens as S. viridis Blatt.& Hallb.nom.illeg., which could not be used.As per the current taxonomy, the species fits in the genus Ledebouria Roth and should be named accordingly.The epithet 'viridis' also could not be used as per ICBN Article 11.4, therefore, the species is renamed as:

Etymology of the epithet
The epithet 'junnarensis' is derived from the locality of the present specimens, falling under the tehsil Junnar in Pune District, Maharashtra State, India.The re-collection of specimens from the original location Khandala was unfruitful and considered to be extirpated from there.
Fruit set rarely.Perianth persistent at fruiting.Seeds ellipsoidal, dark brown, shiny with polygonal reticulation on testa.

Phenology
Plants emerge after the pre-monsoon showers as inflorescence and 2 or 3 leaves in the month of May-June.The leaves grow very fast after the flowering and attain a length of 30-45 cm.Fruits develop and mature in June-July, but the number of mature fruits per plant is very low (about 1-3 fruits).The same flowering period has been observed for the specimens under cultivation.The plants remain dormant from September to mid May in the form of underground bulbs.

Ecology
The population occurs on strong slopes of wellaerated deep soil rich in humus accumulated from grass biomass.

Cytology
The mitotic metaphase cell plates of root tip squash revealed that the cells contain 2n=70 chromosomes.This chromosome number is the highest and not yet reported from any of the Ledebouria species (Image 2).

Leaf Anatomy
The leaf anatomy revealed that the margin of the leaf is single cell in cross section.Epidermal cells are rectangular on both the surfaces.Leaves are amphistomatic; stomata paracytic with two small reniform subsidiary cells parallel to both the guard cells (Image 2).Cataphyll cells contain acicular silica crystals and ellipsoidal starch grains with concentric striations.

Identification keys
For the identification of genera and species found in India the genus and species keys are prepared using the literature and characters of specimens studied.

DISCUSSION
The species is confirmed on the basis of morphological, anatomical, and cytological characters.A comparison in the characteristics of this species with other three species reported from India is given in Table 2.The leaves in Ledebouria junnarensis nom.nov.are 2 or 3 per vegetation period, linear, canaliculate with fleshy tip always producing bulbils and plantlets, upto 45cm long, scape single, inflorescence lax raceme, bracts two, ovary forming a dome, placenta axile, 2 or 3 ovules in each locule, chromosomes 2n=70, smaller silica crystals and starch grains concentric striations.L. revoluta has larger silica crystals and irregularly shaped large starch grains without concentric striations.The epidermal cells and stomata are larger than that of L. revoluta, therefore, stomatal density is relatively less in the species under study.The stomatal index is also less.Floral parts, viz., scale, pedicel, perianth and stamens are pink/purple in L. junnarensis, whereas they are pale greenish-pink in L. revoluta.The flowers are smaller in later species.

Cytology
The mitotic metaphase cell plates of root tip squash revealed that the plants contain 2n=70 chromosomes.This chromosome number is the highest and not yet reported from any of the Ledebouria species.Rao (1956) had identified three cytological variablesdiploids, triploids and tetraploids of L. revoluta (S. indica) populations into two natural morphological groups.
One morphological group comprises diploids (2n=30) and tetraploids (2n=60); while the other morphological group of triploids (2n=45).Unfortunately, detailed morphological characterization is not clear for triploid as well as the tetraploid forms.More importantly, the triploid form studied by him was collected from the Coromandel Coast from Machilipatanam (in Andhra Pradesh) to Chennai (Madras) while, prevalence of diploids and tetraploid forms is in Maharashtra, particularly in the Mumbai area (Rao 1956).Though the triploid form of L. revoluta, which has linear leaves and without or very rarely with seed set, is different in chromosome number, i.e., 2n=45 when compared to L. junnarensis nom.nov.A specimen at Kew -K000802739 collected by Wight in October 1835 from Tamil Nadu, India (RBG Kew 2015) had long linear leaves without scape and bulbils which might be a triploid form of L. revoluta.Comparison of the cytological evidences and present observations of the cytology strongly suggest that the specimen under study is a distinct species and not the triploid form of L. revoluta.The chromosomes recorded here are 2n=70; are first observations and the highest known 2n chromosome number in the genus.Speta (1998a) reported that the genus Ledebouria has species with chromosome numbers 2n=20, 24, 26, 28, 30, 32, 34, 36, 44, 45, 46, 54, 58, 60, and 66.The observations of the present study also confirm a larger number of metaphase chromosomes in the genus, further supporting the distinctiveness of the taxon.L. junnarensis also shows affinities with F. hohenackeri which has linear leaves and axile placentation; however they differ in having a dome shaped, stipitate, green ovary and stellate non-blue flowers with cup shaped perigone and the chromosome number is also different; 2n=10 in later species (Rao 1954).
Key for the genera of the subfamily Hyacinthoideae considering the species reported from India.

Phenology
During the present study, the flowering is observed from May to June and later fruiting has been recorded.The same flowering & fruiting period has been observed for the specimens under cultivation.The flowering period reported in the literature as March (Blatter & Hallberg 1921;Santapau 1958;Deb & Dasgupta 1975, 1981) and from September to March (Sharma et al. 1996;Mishra & Singh 2001) is found to be dubious as later workers referred to the type specimen, which was reported to flower in March.Moreover, the plants are monsoon ephemeral and become dormant by September.The flowering in the month of March reported for the type specimen may be due to drying up after collection and then warm weather with watering can induce flowering due to breaking of the dormancy of the bulb.A similar phenomenon was also observed in a plant under cultivation.

IUCN ASSESSMENT
The present location is on the hill slopes of the Sahyadri in the northern Western Ghats, in Pune District.The present locality is a steep slope and surrounded by privately owned lands on the upper plateau as well as the lower slope.Landslides due to tree felling, and heavy grazing are the major threats.The major population is along the cut edges of the road approaching Durgawadi Plateau, and road widening is a major threat to the population.Thus, deterioration of the habitat is a critical problem to the present population of the species.
On communication with local people, it came to light that the plant is not presently being used for any purpose.As locals do not find any application, they are not interested in conservation of the plant at present.As the plants are amidst the grasses, grazing by goats and cattle cause elimination of terminal bulbils, which are a major means of multiplication.Therefore, grazing is also one of the most potent causes of destruction of the population.
The type locality 'Khandala' around 18 0 0'N & 73 0 23'E and with an elevation of about 536m is situated about 60km away towards the southwest of the present location.Repeated attempts by taxonomists in the past and by the present authors to re-collect the species from the type locality have been unsuccessful.While working for about 12 years from 1940 to 1951 on the Flora of Khandala, Santapau was unable to collect any specimen of the species and reported it only on the authority of first description (Santapau 1958), and the same was followed by subsequent researchers (Deb & Dasgupta 1981;Nayar & Sastry 1987;Karthikeyan et al. 1989;Sharma et al. 1996).Based on these reports the taxon was declared as 'possibly extinct'.Thus, it is clear that, one location is lost forever.The AOO of this location is not known.
An important reason behind the elimination of plants from their type location may be rapid anthropogenic developmental activities that took place at the hill station 'Khandala'.To understand this, one must consider the following facts: (i) the year of reporting of the specimen is 1921; (ii) the first attempt to re-collect the specimen by Santapau, from 1940 to 1951 was about 30 years after the first description; (iii) another 30 or more years are taken for subsequent unfruitful attempts to recollect the specimen by other taxonomists; and (iv) very rapid developmental activities and construction of farm houses and residential complexes.The present situation is that there is hardly any space for natural populations of plants in the hill town and the slopes of the valleys are almost unapproachable.
Information collected during this study indicates that L. junnarensis nom.nov.fulfils the necessary criteria to place it in the 'Critically Endangered' category (IUCN 2015) as it faces a high risk of extinction.The EOO is approximately 60km 2 considering its type locality and the new locality under present study.It is very important that, specimens could not be found at the type locality, thus the original location is lost for the taxon.This fact has reduced the EOO to almost 50% or more.Even if both the locations are considered for EOO, it is less than 100km 2 and the species satisfies the condition B1.
The AOO of the taxon at the present location is about 0.52km 2 , which is far less than 10km 2 .Considering the lost population at type location there is sure reduction in the AOO also.Therefore it satisfies condition B2a.
The EOO and AOO have declined and the quality of habitat is showing a continuous decline.These facts satisfy the criteria for the categorization of the taxon as Critical Endangered (CR) under criterion B of IUCN.
It is evident from the literature that the species was spread over a larger area but now reduced to only one location.This location also has a small population of <1000 individuals.The emphasis should also be given to the mode of reproduction through bulbils at the tip of each leaf.A single plantlet results from each bulbil.Considering the average leaves per vegetation period 3-4 (av.3.5) each plant can produce 3.5 plantlets per vegetation period.But this is not actually observed; instead the population shows more or less a stable number of individuals.The main reason behind this phenomenon is the grazing by cattle, which eliminate leaf bulbils while grazing and the plantlets could not be produced.
The present population of the species is subjected to heavy grazing, trampling by grazers and cattle.The threat of road widening and landslides are added ones.The lack of dispersal of plantlets due to the mode of reproduction is very important and also a threat to the species.The locals are unaware about the species and so are not interested in conserving it in the absence of any usage value to it.It is also noted by the authors that the visitors from neighbouring areas are increasing yearly for pilgrimage and tourist activities at Durgawadi Plateau.The transport facilities are likely to be improved in the very near future, which is primarily in the form of road widening.Thus, the fate of the species is unknown.The above measured and quantified facts and observations clearly indicate that the species fulfils both the conditions B1 & B2 of Criterion B to qualify for CR category.Therefore, the status of Ledebouria junnarensis S.S. Rahangdale & S.R. Rahangdale is assigned as Critically Endangered B1ab(iii)+2ab(iii) with justification (Appendix 1).

CONCLUSION
The taxon which was declared as 'possibly extinct' after unfruitful attempts to re-collect it from its type location and other locations by several taxonomists has now been rediscovered after about 85 years.The taxon has been studied with respect to vegetative and floral anatomy, cytology and the identity has been confirmed.The taxon is assigned to the new name in the present systematics framework as, Ledebouria junnarensis S.S. Rahangdale & S.R. Rahangdale nom.nov.
It is also evaluated for threat status as per IUCN criteria and guidelines Version 6.2 and categorized as 'Critically Endangered' vide condition B1ab(iii)+2ab(iii).The population at the type location is lost due to anthropogenic activities and the present location is supporting the only surviving population of the species.This location is also under threat of grazing, habitat degradation, landslide and road widening, beside the propagation restriction of formation of plantlets from the tip bulbil and very rarely through seeds.Therefore, in situ, as well as ex situ conservation measures should be prioritized.It can be introduced in other similar and safer habitats in the Western Ghats after evaluation as per the guidelines of the IUCN Reintroduction Specialist Group.It is evident from the experience of authors that the plant thrives well under pot/bed cultivation and also multiplies.It is a beautiful ephemeral whose purple/ pink flowers and blotched leaves have an ornamental potential.Therefore, ex situ conservation is also possible.

Image 1 .
Ledebouria junnarensis S.S. Rahangdale & S.R. Rahangdale.A -Habit; B -Single plant at full growth on graph paper; C -Canaliculate leaf with fleshy tip; D -Bracts; E -Cross section of bulb showing scale leaves of 2-3 vegetation period with single scape per vegetation period; F -Leaf margin; G -Cross section of leaf margin; H -Flower; I -Carpel; J -TS & VS of ovary; K -Fruits with persistent perianth; L -Multiplication through bulbils produced at leaf tips (series of developmental stages).(Photographs and diagrams by S.R. Rahangdale) The Pimparwadi-Inglun Ghat (Durgawadi), is the location of the single present population of the species.The geographic position of the location is 19 0 11'42.10"N& 73 0 43'43.69"E,with an elevation range of 990-1095 m.The present population is of about 680 individuals (in 2013) in a very small area (AOO) of 0.52km 2 (Image 3) along the grassy hill slope between 990-1095 m in association with Chlorophytum tuberosum Baker, Crinum latifolium L. var.latifolium, Cyanotis tuberosa (Roxb.)J.A. & J.H. Schult., Euphorbia fusiformis Buch-Ham.ex D. Don, Habenaria grandifloriformis Blatt.& McCann, Iphigenia magnifica Ansari & Rolla Rao and Iphigenia pallida Baker.The species has been recorded for the first time from the region.Extensive surveys by the authors from 2005 to 2014 comprising Maharashtra State including the Western Ghats, especially Pune District and type Locality 'Khandala' did not result in the observation of specimens similar to the present one.The population is more or less stable and maintained by the plantlet formation by bulbils and rarely by seeds.

Table 2 . Comparison of the characters of four taxa of Hyacinthoidae reported from India.
NA -Data not availableRediscovery